The present invention relates to a method of determining proBNP or fragments thereof in mammals.
Heart diseases play an important part not only in humans, but also animals, in particular pets, such as dogs or cats, are afflicted with these diseases. Studies have shown that, e.g., each tenth canine heart has a functional impairment. The heart diseases occurring concern, for instance, the cardiac valves and the cardiac muscle. Since at first the heart is capable of compensating functional impairment by working harder, such a disease in most cases remains hidden, with the consequence that the state of the heart will deteriorate due to the increased load on the heart. The symptoms resulting from heart diseases, such as fatigue, circulatory insufficiency, languor, can mostly be recognized when the pet's heart is no longer able to compensate the weakness. In such a case, the heart disease has already progressed so much that complete curing is hardly possible any more.
As a rule, chronic cardiac valve and cardiac muscle changes are not curable, yet by the use of medicaments, the further progress of the heart disease can be slowed down. Therefore, an early diagnosis must be made for the occurring heart diseases. By way of routine, mainly physical methods are used for this purpose, such as auscultation of the heart sounds, the recording of an electrocardiogram, X-ray and ultrasonic examinations. These examination methods mainly have the disadvantage that they can be carried out only when already visible or audible defects can be directly recognized on the heart. Furthermore, physical examination methods require suitable and, as a rule, expensive devices in order to carry out a respective diagnosis.
The heart diseases most frequently occurring in dogs, e.g., are heart decompensation and dilated cardiomyopathy, which mainly afflict big animals. Dilated cardiomyopathy is a heart disease which causes an enlargement of the ventricles of the heart with normal wall thickness, such enlargement quickly causing cardiac insufficiency in the afflicted animal. By admixing taurine to the feed, the risk of falling ill with dilated cardiomyopathy could be reduced significantly. In an illness related to dilated cardiomyopathy, the restrictive cardiomyopathy which frequently is found in older cats, a continuous decrease in the heart function with a reduced ability for pumping can be observed. The heart disease most frequently occurring in cats is hypertrophic cardiomyopathy. This disease of the cardiac muscle causes thickening of the heart wall and a resultant reduced ability to fill the ventricles of the heart with blood. This leads to an accumulation of blood in the left ventricle and to a greatly reduced amount of blood being pumped through the body.
In many heart diseases, such as, e.g., cardiac insufficiency, dilated cardiomyopathy, hypertrophic cardiomyopathy, left-ventricular hypertrophy and dysfunction, a peptide hormone, the so-called BNP (brain natriuretic peptide) is secreted. This hormone causes the excretion of liquid via the kidneys and thus regulates the cardiovascular system. Since this peptide is produced in the heart and is increasingly produced in case of an overstress and congestion of the heart, determining the BNP level in blood is a suitable means for evaluating cardiac insufficiency.
BNP as well as other natriuretic peptides play an important part in regulating the water balance and the blood pressure. If the cardiac wall is dilated, it secrets BNP in increasing amounts, causing an excretion of sodium and liquid via the kidneys and a dilation of the blood vessels, which in sum can lower the blood pressure and the filling level of the heart. BNP is synthesized by the cells of the cardiac muscle as proBNP which finally is cleaved into n-terminal proBNP and BNP. Both parts of the BNP are delivered to the blood and can be determined therein.
Cardiac diseases in animals are, inter alia, dealt with in the following pertinent publications: Bright J M and Cali J V, J Am Vet Med Assoc 2000, 216:1110-4; Guglielmini C, Vet Res Commun 2003, 27 Suppl 1:555; Boswood A et al., J Small Anim Pract 2003, 44:104-8; Takemura N et al., J Vet Med Sci 2003, 65:1265-7; MacDonald K A et al., J Vet Intern Med 2003, 17:172-7; Greco D S et al., Can Vet J 2003, 44:293-7; Monnet E et al., J Am Vet Med Assoc 1997, 211:569-72; Hamlin R L et al., J Vet Intern Med 1996, 10:85-7; Gaschen L et al., J Vet Intern Med 1999, 13:346-56.
A large number of methods are already known in the prior art which assist in the detection of human proBNP or the fragments thereof, respectively, in the serum of an individual. By way of example, here EP 0 648 228 B1, WO 03/87819 and FR 2 843 396 should be mentioned.
In US 2004/0018577, an immunoassay is disclosed which comprises at least three antibodies which all are capable of binding to different epitopes of an analyte. The analytes to be detected particularly concern the detection of markers regarding heart diseases, wherein i.a. also BNP and proBNP can be detected.
Biondo A. W. et al. (Vet. Pathol. 2003, 40(5):501-506) describe a method of detecting ANP and BNP in cats by means of polyclonal antibodies which are directed against a peptide of the ANP which comprises the amino acids 1 to 28, and against a peptide which comprises the amino acids 43 to 56 of proBNP, respectively.
In EP 1 016 867 A1, an immunoassay is described for the detection of preproBNP in mammals. There, antibodies are used which are directed against peptides comprising the amino acids 27 to 102, 73 to 102 and 27 to 64 of human BNP.
Jortani S. A. et al. (Clin. Chem. 2003, 50(2):265-278) describe the use of BNP and its prepro- and pro-forms as possible markers for heart diseases. In this article, no preferred peptide regions of BNP are mentioned which would be suitable for detecting heart diseases in dogs and cats.
In WO 2000/35951 several peptides are disclosed against which antibodies can be prepared, which are suitable in a method for diagnosing heart diseases. Three peptides comprising the amino acids 1 to 13, 37 to 49, and 65 to 76 of human Nt-pro-BNP protein are disclosed there, which may also be used for preparing antibodies that are directed against these peptides.
Moreover, several test kits for detecting human proBNP or the fragments thereof, respectively, are commercially available (e.g. from Roche and Biomedica). Nevertheless, there is no known method with whose assistance specifically proBNP in animal samples can be determined. Therefore, and because of the costly and complicated physical examinations of animals it is an object of the present invention to provide suitable means for determining proBNP or the fragments thereof, respectively.